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Title:
APPARATUS FOR AND METHODS OF REMOVING FLUID FROM A CELL CULTURE
Document Type and Number:
WIPO Patent Application WO/2019/071297
Kind Code:
A1
Abstract:
An apparatus for removing fluid from a cell culture comprising the fluid and one or more multicellular spheroids, the apparatus comprising: a body having an conduit terminating at an opening; and a filter extending across the opening, the filter having a pore size of less than the diameter of the one or more multicellular spheroids so as to prevent the one or more multicellular spheroids from passing through the filter and into the conduit whilst allowing the fluid to pass through the filter and into the conduit.

Inventors:
ST JOHN JAMES (AU)
VIAL MARIE-LAURE (AU)
CHEN MO (AU)
Application Number:
PCT/AU2018/051083
Publication Date:
April 18, 2019
Filing Date:
October 08, 2018
Export Citation:
Click for automatic bibliography generation   Help
Assignee:
UNIV GRIFFITH (AU)
International Classes:
B01L3/02; C12M1/12; C12M3/06; C12N5/00
Foreign References:
US20120252008A12012-10-04
US20120164750A12012-06-28
EP2806020A12014-11-26
EP0588564A11994-03-23
US20170074758A12017-03-16
Other References:
CRIBB, B. ET AL.: "A simple filter system for processing small or transparent specimens", JOURNAL OF MICROSCOPY, vol. 17, no. 3, 1994, pages 83 - 86, XP055592469, ISSN: 0022-2720, DOI: 10.1111/j.1365-2818.1994.tb03431.x
CANCER STEM CELL TUMORSPHERE FORMATION PROTOCOL SIGMA ALDRICH, 14 September 2017 (2017-09-14), Retrieved from the Internet
Attorney, Agent or Firm:
FB RICE (AU)
Download PDF:
Claims:
CLAIMS:

1. An apparatus for removing fluid from a cell culture comprising the fluid and one or more multicellular spheroids, the apparatus comprising:

a body having an conduit terminating at an opening; and

a filter extending across the opening, the filter having a pore size of less than the diameter of the one or more multicellular spheroids so as to prevent the one or more multicellular spheroids from passing through the filter and into the conduit whilst allowing the fluid to pass through the filter and into the conduit.

2. The apparatus of claim 1, wherein the filter extends at least level to the opening.

3. The apparatus of claims 1 or 2, wherein the filter is disposed within the conduit, and wherein a perimeter of the filter is in contact with an interior wall of the conduit.

4. The apparatus of claims 3, wherein the perimeter of the filter is in contact with an internal annulus of the opening.

5. The apparatus of claims 3 or 4, wherein the filter extends outside of the conduit through the opening.

6. The apparatus of claims 1 or 2, wherein the filter is disposed on the outside of the conduit overlapping the opening.

7. The apparatus of any one of the preceding claims, wherein the filter is configured to prevent particles having a diameter greater than 60 μιη from entering the conduit via the opening.

8. The apparatus of any one of the preceding claims, wherein the filter is configured to prevent particles having a diameter greater than 40 μιη from entering the conduit via the opening.

9. The apparatus of any one of the preceding claims, wherein the filter is configured to prevent particles having a diameter greater than 10 μιη, or greater than 20 μι¾ or greater than 30 μιη, or greater than 50 μιη, or greater than 70 μιη, or greater than 80 μιη, or greater than 90 μιη, or greater than 100 μιη from entering the conduit via the opening.

10. The apparatus of any one of the preceding claims, wherein the filter comprises a nylon mesh.

11. The apparatus of any one of the preceding claims, further comprising a pump configured to draw fluid into the conduit via the opening.

12. The apparatus of any one of the preceding claims, wherein the body is a pipette tip.

13. The apparatus of any one of the preceding claims, wherein the body is disposable.

14. The apparatus of any one of the preceding claims, wherein the body is made from plastic.

15. The apparatus of any one of the preceding claims, wherein the apparatus is a pipette.

16. The apparatus of any one of the preceding claims, wherein the fluid comprises one or more of a cell medium, a reagent, and a buffer.

17. The apparatus of any one of the preceding claims, wherein the one or more multicellular spheroids comprises one or more of the following:

a) stem cells;

b) blastocysts;

c) differentiated cells; and

d) partially differentiated cells.

18. The apparatus of any one of the preceding claims, wherein the one or more multicellular spheroids comprises one or more olfactory ensheathing cells (OECs), or one or more cancer cells, or one or more progenitor cells, or one or more stem cells.

19. The apparatus of any one of the preceding claims, wherein the filter has a thickness of between 20 μηι and 200 μπι.

20. An automated liquid handling platform, comprising one or more apparatus according to any one of claims 1 to 19.

21. A high-throughput liquid handling platform, comprising one or more apparatus according to any one of claims 1 to 19.

22. A method of removing fluid from a cell culture comprising the fluid and one or more multicellular spheroids, the method comprising:

drawing off the fluid from the cell culture through a filter, the filter having a pore size of less than the diameter of the one or more multicellular spheroids so as to prevent the one or more multicellular spheroids from passing through the filter whilst allowing the fluid to pass through the filter.

23. The method of claim 22, further comprising detaching one or more

multicellular spheroids which have become attached to the filter during the drawing off.

24. The method of claims 22 or 23, wherein the pore size of the filter is less than or equal to 100 μιτι, less than or equal to 90 μιτι, less than or equal to 80 μιτι, less than or equal to 70 μιτι, or less than or equal to 60 μιτι, less than or equal to 50 μιτι, or less than or equal to 40 μιτι, less than or equal to 30 μιτι, or less than or equal to 20 μηι.

25. The method of claim 22 or 23, wherein the pore size of the filter is between 20 μηι and 60 μιη.

26. The method of claim 25, wherein the pore size of the filter is 40 μηι.

27. The method of any one of claims 22 to 26, wherein the fluid comprises one or more of a cell medium, a reagent, and a buffer.

28. The method of any one of claims 22 to 27, wherein the one or more multicellular spheroids comprises one or more of the following:

a) stem cells;

b) blastocysts;

c) differentiated cells; and

d) partially differentiated cells.

29. The method of any one of claims 22 to 28, wherein the one or more multicellular spheroids comprises one or more olfactory ensheathing cells (OECs), or one or more cancer cells, or one or more progenitor cells, or one or more stem cells.

30. The method of any one of claims 22 to 29, wherein the filter has a thickness of between 20 μιη and 200 μιη.

31. The method of any one of claims 22 to 30, wherein the fluid is drawn off from the cell culture via an apparatus according to any one of claims 1 to 19 or via an automated liquid handling platform according to claim 20, or via a high-throughput liquid handling platform according to claim 21.

32. The steps, features, integers, compositions and/or compounds disclosed herein or indicated in the specification of this application individually or collectively, and any and all combinations of two or more of said steps or features.

Description:
"Apparatus for and methods of removing fluid from a cell culture" Technical Field

[0001] Embodiments of the present disclosure relate to apparatus for and methods of removing fluid from cell cultures.

Background

[0002] Three-dimensional (3D) cell culture systems, which can more closely mimic the in vivo state than conventional two-dimensional (2D) cell cultures, are used in a broad spectrum of applications including drug discovery, stem cell-based therapies and developmental biology.

[0003] Currently, 3D cell cultures are produced and manipulated using cell analysis and cell manipulation techniques (e.g., including liquid change or washing techniques) originally developed for 2D cell culture systems. Applying such techniques to 3D cell cultures, particularly techniques requiring multiple wash steps, can be challenging. Due to the nature of cell growth in 3D cell cultures, spheroids and other 3D cell structures tend to have low adherence to surfaces of vessels in which they are being cultured. This means that using conventional liquid change techniques and apparatus, 3D structures can be easily sucked up out of the culture vessel along with the liquid. Other techniques used for media change and washing of 3D cell cultures include 50/50 media exchange, centrifugation and free settling techniques. However, these techniques are time-consuming and can damage the morphology of the spheroids or even lead to destruction of the spheroids during transfer from one device to another.

[0004] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims. Summary

[0005] The present disclosure addresses issues associated with handling 3D cell cultures. Particularly, the inventors have developed apparatus and methods for removing liquid from 3D cell cultures that can prevent or at least mitigate against damage or destruction of 3D multicellular spheroids present in the 3D cell cultures. For example, an apparatus has been developed which comprises a filtration mechanism which allows liquid to flow into the apparatus but prevents spheroids having a diameter greater than a predetermined size from entering the apparatus. The apparatus and methods may provide for fast and efficient liquid transfer and replacement in 3D cell cultures, without the requirement of expensive machines and time-consuming laboratory techniques.

[0006] Embodiments of the present disclosure may allow for complete media change in 3D cell cultures and may facilitate the washing steps of common imaging techniques (e.g. immunofluorescence staining), without losing or damaging multicellular spheroids. Embodiments of the present disclosure may be suitable for use in washing out drugs during drug screening and may be used in association with automated liquid handling platforms such as the Agilent (RTM) Bravo (RTM). Embodiments of the present disclosure may provide for easier handling of liquids, in particular for 3D cell culturing, immunofluorescence assays, drug discovery using 3D multicellular spheroids and blastocyst handling.

[0007] According to a first aspect of the present disclosure, there is provided an apparatus for removing fluid from a cell culture comprising the fluid and one or more multicellular spheroids, the apparatus comprising: a body having a conduit terminating at an opening; and a filter extending across the opening, the filter having a pore size of less than the diameter of the one or more multicellular spheroids so as to prevent the one or more multicellular spheroids from passing through the filter and into the conduit whilst allowing the fluid to pass through the filter and into the conduit. The cell culture may be a 3D cell culture. Thus, the apparatus may be for removing fluid from a 3D cell culture. [0008] The filter may extend at least level to the opening, either on the inside or on the outside of the conduit. The filter may be disposed within the conduit. When the filter is disposed within the conduit, the perimeter of the filter may be in contact with an internal annulus of the opening. The perimeter of the filter may be in contact with an interior wall of the conduit. Alternatively or additionally, the filter may extend outside of the conduit through the opening.

[0009] In some embodiments, at least part of the filter may be disposed on the outside of the conduit overlapping the opening. A surface of the filter may be flush with the opening. Alternatively, there may be a gap between the filter and the opening. In any case, the filter is provided to prevent the one or more multicellular spheroids from passing through the filter and into the conduit whilst allowing the fluid to pass through the filter and into the conduit.

[0010] It will be appreciated that, in any embodiment disclosed herein, the filter may prevent the one or more multicellular spheroids from passing through the filter and into the conduit by preventing the one or more multicellular spheroids from entering into and/or being retained in the filter material. Thus, the filter may be provided so as to prevent one or more multicellular spheroids from entering into the filter, where they may become trapped.

[0011] The filter may be configured to prevent particles having a diameter greater than 60 μιη or greater than 40 μιη or greater than 20 μιη from entering the conduit via the opening.

[0012] The filter may comprise a mesh, The mesh may have a uniform porosity. The mesh may be made from nylon or gauze, It will be appreciated that other suitable materials may be used.

[0013] A pump may be provided to draw fluid into the conduit via the opening. The pump may be in the form of a syringe, a pipette or other pumping means. The pump may generate a negative pressure in the conduit, so as to draw liquid into the conduit via the opening.

[0014] The body may be elongate. In some embodiments, the body may be a pipette tip.

[0015] The body may be disposable. Additionally, or alternatively, the body may made from plastic.

[0016] In some embodiments, the apparatus may be a pipette or pipette tip which may or may not be disposable.

[0017] The fluid may comprise one or more of a cell medium, a reagent, and a buffer.

[0018] The one or more multicellular spheroids may comprise one or more differentiated or partially differentiated cells. The one or more multicellular spheroids may comprise stem cells and/or one or more blastocysts. The one or more multicellular cells may comprise one or more olfactory ensheathing cells (OECs) , and/or one or more cancer cells, and/or one or more progenitor cells, and/or one or more stem cells. The one or more multicellular spheroids may comprise a single cell type or multiple cell types. The multiple cell types may include one or more of the above cell types.

[0019] The apparatus may be incorporated into an automated liquid handling platform, the automated liquid handling platform being configured to automatically remove fluid from a cell culture comprising the fluid and one or more multicellular spheroids. Additionally or alternatively, the apparatus may be incorporated into a high throughput liquid handling apparatus being configured to remove fluid from a cell culture comprising the fluid and one or more multicellular spheroids. An example of an automated liquid handling platform is the Agilent (RTM) Bravo (RTM). An example of a high throughput liquid handling platform is the Agilent (RTM) Bravo (RTM). [0020] According to another aspect of the present disclosure, there is provided a pipette tip, comprising: a conduit terminating at an open tip end; and a filter disposed within the internal conduit and having an exterior perimeter in contact with an interior wall of the internal conduit, the filter extending at least level with the open tip end of the pipette tip.

[0021] The filter may protrude through the open tip end of the pipette tip.

[0022] The external perimeter of the filter may be in contact with an internal annulus of the open tip end.

[0023] The filter may be configured to prevent particles having a diameter greater than 60 μιη or greater than 40 μιη or greater than 20 μιη from entering the conduit via the opening.

[0024] The filter may comprise a mesh. The mesh may have a uniform porosity. The mesh may be made from nylon or gauze.

[0025] The pipette tip may be disposable. The pipette tip may be made from plastic.

[0026] The filter may have a thickness of between 20 μιη and 200 μιη.

[0027] The filter may have a thickness greater than 20 μιη, or greater than 30 μιη, or greater than 40 μιη, or greater than 50 μιη, or greater than 60 μιη, or greater than 70 μι¾ or greater than 80 μιη, or greater than 90 μιη, or greater than 100 μιη, or greater than 110 μιη, or greater than 120 μιη, or greater than 140 μιη, or greater than 160 μιη, or greater than 170 μιη, or greater than 180 μιη, or greater than 190 μιη.

[0028] The filter may have a thickness less than 30 μιη, or less than 40 μιη, or less than 50 μιη, or less than 60 μιη, or less than 70 μιη, or less than 80 μιη, or less than 90 μι¾ or less than 100 μιη, or less than 110 μιη, or less than 120 μιη, or less than 140 μι¾ or less than 160 μιη, or less than 170 μιη, or less than 180 μιη, or less than 190 μιη. [0029] According to another aspect of the disclosure, there is provided a pipette comprising an apparatus or pipette tip as described above.

[0030] According to another aspect of the disclosure, there is provided a method of removing fluid from a cell culture comprising the fluid and one or more multicellular spheroids, the method comprising: drawing off the fluid from the cell culture through a filter, the filter having a pore size of less than the diameter of the one or more multicellular spheroids so as to prevent the one or more multicellular spheroids from passing through the filter whilst allowing the fluid to pass through the filter. The cell culture may be a 3D cell culture. Thus, the method may be a method of removing fluid from a 3D cell culture.

[0031] The method may further comprise detaching one or more multicellular spheroids which have become attached to the filter during the drawing off. The detaching may be instigated by altering the pressure in the conduit so as to reduce or eliminate any suction maintaining attachment of the multicellular spheroids to the filter.

[0032] The method disclosed herein may particularly be employed as an automated method of removing fluid from a cell culture comprising the fluid and one or more multicellular spheroids, and/or as a high throughput method of removing fluid from a cell culture comprising the fluid and one or more multicellular spheroids.

[0033] The pore size of the filter may be less than or equal to 60 μιη, or less than or equal to 40 μιη, or less than or equal to 20 μιη. In some embodiments, the pore size of the filter is between 20 μιη and 60 μιη. In some embodiments, the pore size of the filter is 40 μιη.

[0034] The fluid may comprise one or more of a cell medium, a reagent, and a buffer.

[0035] The one or more multicellular spheroids may comprise one or more stem cells and/or one or more blastocysts. The one or more stem cells may comprise one or more olfactory ensheathing cells (OECs), and/or one or more cancer cells, and/or one or more progenitor cells, and/or one or more stem cells.

[0036] The fluid may be drawn off from the cell culture via an apparatus or pipette tip as described above. Equally the method described herein may be performed by an apparatus, pipette or pipette tip as described herein.

[0037] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Brief Description of Drawings

[0038] By way of example only, embodiments of the present disclosure are now described with reference to the Figures in which:

[0039] Figure 1 is a schematic illustration of an apparatus according to an

embodiment of the disclosure;

[0040] Figure 2 is a schematic illustration of a variation of the apparatus according to an embodiment of the disclosure;

[0041] Figure 3 is a schematic illustration of a variation of the apparatus according to an embodiment of the disclosure;

[0042] Figure 4 is a schematic illustration of a variation of the apparatus according to an embodiment of the disclosure;

[0043] Figures 5 and 6 are schematic illustrations showing the apparatus of Figure 1 in use; [0044] Figure 7 shows images taken of multicellular spheroids before and after media change using each of a conventional 50/50 media change technique and the apparatus illustrated in Figure 1 ;

[0045] Figure 8 shows the percentage of spheroids remaining after media change using each of a conventional 50/50 media change technique and the apparatus illustrated in Figure 1 ;

[0046] Figure 9 shows images taken by the Olympus (RTM) confocal FV1000 microscope of a co-culture of astrocytes detected by anti-GFAP and SlOOp-DsRed Schwann cells;

[0047] Figure 10 shows images taken by an Olympus (RTM) confocal FV1000 microscope of co-culture of astrocytes detected by anti-GFAP and SlOOp-DsRed olfactory ensheathing cells (OEC);

[0048] Figure 11 shows images of fixed 3D OEC spheroids stained with Hoechst and CellMask Deep Red using a conventional centrifugation technique and the apparatus illustrated in Figure 1 ;

[0049] Figure 12 illustrates the comparative sphericity of 3D structures after immunofluorescence assay using a conventional centrifugation technique and the apparatus illustrated in Figure 1;

[0050] Figure 13 illustrates the comparative percentage of spheroids remaining after immunofluorescence assay using a conventional centrifugation technique and the apparatus illustrated in Figure 1;

[0051] Figure 14 shows representative images of unfixed 3D OEC spheroids stained with Hoechst and CellMask Deep Red using a conventional centrifugation technique and the apparatus illustrated in Figure 1; [0052] Figure 15 illustrates the comparative sphericity of 3D structures after immunofluorescence assay using a conventional centrifugation technique and the apparatus illustrated in Figure 1; and

[0053] Figure 16 illustrates the comparative percentage of spheroids remaining after immunofluorescence assay using a conventional centrifugation technique and the apparatus illustrated in Figure 1

[0054] Figure 17 shows representative images of fixed 3D pancreatic cancer cell (BxPC-3) spheroids stained with Hoechst and Alexa Fluor™ 488 Phalloidin using the apparatus illustrated in Figure 1; and

[0055] Figure 18 shows representative images of fixed 3D neural progenitor cell (ReNcell VM) spheroids stained with Hoechst and Alexa Fluor™ 488 Phalloidin using the apparatus illustrated in Figure 1.

Description of Embodiments

[0056] Figure 1 is a schematic illustration of an apparatus 10 for removing fluid from a cell culture comprising one or more multicellular spheroids. The apparatus comprises a body 12 having a conduit 14 terminating in an opening 16, as well as a filter 17 which extends across the opening 16. The body 12 may be elongate. As will be described in more detail below, the filter is configured to allow fluid 15 to pass through into the conduit 16 via the opening, whilst preventing particles having a diameter greater than a predetermined size from passing into the conduit 16. The filter may be in the form of a thin mesh. The filter may have a thickness in the region of between 20 μιη and 200 μιη. The apparatus 10 may taper in size towards the opening 16 in order to increase the surface tension at the tip whilst maximising the volume within the conduit 14.

[0057] The apparatus 10 may further comprise or be connectable to a device (not shown) for drawing fluid 15 into and/or out of the conduit 14 via the opening 16 as denoted by the double-ended arrow in Figure 1. Such a device may, for example, be in the form of a suction pump, a syringe, a pipette or similar device known in the art.

[0058] The filter 17 may be disposed within the conduit 14, its perimeter in contact with the interior wall of the conduit 16. In which case, the perimeter of the filter 17 is preferably in contact with an internal annulus of the opening 16 to ensure that only particles smaller than the predetermined size enter the conduit 14.

[0059] The pore size of the filter 17 may be chosen in dependence of the size of particles that are to be prevented from entering the conduit 16.

[0060] For example, in applications where fluid is to be separated from multicellular spheroids, which typically have a diameter of between 100 μιη and 200 μιη, the pore size of the filter 17 may be chosen to be substantially less than that of the smallest spheroids that are to be prevented from entering the conduit 10.

[0061] In some embodiments, the pore size of the filter 17 may be chosen to be less than or equal to 100 μιη, or less than or equal to 90 μιη, or less than or equal to 80 μιη, or less than or equal to 70 μιη, or less than or equal to 60 μιη, or less than or equal to 50 μιη, or less than or equal to 40 μιη, or less than or equal to 30 μιη or less than or equal to 20 μιη.

[0062] As well as choosing a pore size which is small enough to prevent unwanted particles from entering the conduit 14, it may also be advantageous to define the pore size to maintain an adequate rate of flow of liquid into and out of the conduit 14. By choosing a pore size which achieves the requisite filtration whilst also maximising flow rate through the filter 10, time and energy efficiency of the apparatus 10 can in turn be maximised.

[0063] In some embodiments, the pore size of the filter 17 may be chosen to be greater than 10 μιη, or greater than 20 μιη, or greater than 30 μιη, or greater than 40 μι¾ or greater than 50 μιη, or greater than 60 μιη, or greater than 70 μιη, or greater than 80 μηι, or greater than 90 μηι, or greater than 100 μηι, depending on the size of the particles to be filtered and the flow rate requirements of the apparatus 10.

[0064] The filter 10 may be in the form of a mesh. The mesh may be made from nylon, gauze or other suitable material. The mesh may have a substantially uniform porosity.

[0065] A variation of the embodiment of Figure 1 is shown in Figure 2, where like parts have been denoted with like numbers. Instead of having a filter disposed within the conduit 14, as shown in Figure 1, a filter 18 is provided external to the conduit 14. The filter 18 has a surface that is flush across the opening 16.

[0066] In another variation of the embodiment of Figure 1, as shown in Figure 3, a filter 20 is provided that is external to the conduit 14 and which wraps around the end of the conduit 14 enclosing the opening 16. As with the filter 18 shown in Figure 2, the filter has a surface that is flush across the opening 16.

[0067] In yet a further variation of the embodiment of Figure 1, as shown in Figure 4, a filter 22 is provided that is external to the conduit 14 and that encloses the end of the conduit 14 in a cage-like manner. The filter 22 extends across the opening 16 but does not contact the annulus of the opening 16.

[0068] In other variations, a filter may be provided which extends both inside the conduit 14 and around the body 12.

[0069] In each of the above-described embodiments, a filter extends across the opening 16 of the conduit 14 so as to prevent particles greater than the predetermined size from entering the conduit 14. Thus, the particles may not merely be prevented from travelling through the conduit 14, they may be prevented from entering the conduit 14 at all. [0070] The filter may be attached to the body 12 by any suitable means. It will be appreciated that a filter may be attached to one or a plurality of bodies 12 by any suitable means. For example, one filter may be attached to each of multiple bodies 12 provided in a multichannel pipette apparatus, separately. Alternatively or in addition, one filter may be configured in a multichannel pipette apparatus so as to be attached to each of multiple bodies 12 provided in the multichannel pipette apparatus.

[0071] Figures 5 and 6 illustrate operation of the apparatus in removing fluid 15 from a cell culture 24 comprising the fluid 15 and one or more multicellular spheroids 26.

[0072] The apparatus 10 is lowered into the cell culture 24 such that the opening 16 is submerged in fluid 15.

[0073] A negative pressure is then generated within the conduit 14 (by a pipette, syringe, pump or otherwise) causing the fluid 15 contained in the cell culture 24 to be drawn through the filter 17 and into the conduit 14.

[0074] During the drawing off of the fluid 15 from the cell culture 24, the filter 17 acts as a barrier to prevent the multicellular spheroids 26 present in the cell culture 24 from entering the conduit 14.

[0075] Once the requisite amount of fluid 15 has been drawn out of the cell culture 24 and into the conduit 14, the apparatus 10 may be raised up and away from the cell culture 24 as shown in Figure 6, leaving the multicellular spheroids 26 behind, depleted of the fluid 15 previously present in the cell culture 24.

[0076] It will be appreciated that the drawing off of the fluid through the opening 16 and filter 17 into the conduit 14 may cause one or more of the spheroids 26 to attach to the filter 17 by suction. To ensure that substantially all of the spheroids 26 are left in the cell culture 24 after the apparatus 10 has been removed from the cell culture 24, the negative pressure in the conduit is preferably reduced or removed so as to allow the spheroids 26 to detach from the filter 17 and fall back into the cell culture 26. It will be appreciated however that any reduction or removal of the negative pressure within the conduit should not result in the fluid 15 falling out of the conduit and back into the cell culture 26.

[0077] The cell culture 24 may comprise any cell culture media and any cell culture media components (e.g., growth factors, amino acids, salts, buffers, etc.) suitable for culturing the multicellular spheroids 26.

[0078] As indicated herein, the cell culture 24 may comprise one or more drugs or drug candidates being tested for an effect on the multicellular spheroids 26.

Alternatively or in addition, the cell culture 24 may comprise one or more agents to assist in the analysis of the multicellular spheroids (e.g., including any one or more detectable labels, such as a dye, an antibody, a fluorescent particle, a magnetic particle, or any other suitable detectable label).

[0079] The cell culture fluid may be removed using the apparatus or methods disclosed herein and replaced with any other suitable fluid (e.g., fresh cell culture media, wash buffer, etc.).

[0080] The multicellular spheroids 26 may comprise a plurality of any cells. The cells may be the same or may be different.

[0081] The multicellular spheroids may be cultured for any intended purpose, including in preparation for implantation into a recipient. For example, the

multicellular spheroids 26 may comprise one or more stem cells for implantation into a recipient. Alternatively or additionally, the multicellular spheroids may comprise one or more differentiated or partially differentiated cells for implantation into a recipient. Alternatively or in addition, the multicellular spheroids 26 may comprise one or more blastocysts (which may be at any stage of development) for implantation into a recipient. The recipient may be a mammal, such as a human. The advantages of the apparatus and methods disclosed herein are particularly beneficial when the apparatus is employed in such applications. For example, at least the maintenance of the spherical form of the multicellular spheroids 26, the reduced loss of viable multicellular spheroids 26 during handling, and the reduced experimental handling time are particularly relevant advantages in such applications.

[0082] Examples

Prototype apparatus

[0083] Removal of liquid medium from cell culture containing multicellular spheroids is needed for several different processes including refreshing culture medium, adding specific reagents for assays, and replacing with fixative solution. To test the suitability of using the apparatus described with reference to Figures 1 to 6, herein referred to as "3D-tips", a prototype was assembled in the following manner.

[0084] First, the mesh from a Falcon (RTM) 40 μιη cell strainer (#352340) was cut to approximately 7 mm wide and 15 mm long. A Corning® gel-loading pipette tip (#CLS4884) was cut to a length of approximately 16 mm. The 7 mm x 15 mm mesh was then wrapped around the point of an intact Corning® gel-loading pipette tip (#CLS4884) and inserted inside the Corning® gel-loading pipette tip (#CLS4884) previously cut. Finally, the point of the tip was trimmed and the 3D-tip was then ready for use.

Example 1 - Comparison between the 3D-tip and a normal tip for media exchange

[0085] To test the prototype 3D-tip, the effect of removal of liquid medium from a cell culture containing multicellular spheroids using both conventional methods and the novel 3D-tip were compared.

[0086] Immortalized mouse olfactory ensheathing cells (OECs) were generated from olfactory bulb ensheathing glia of GFP-expressing mice (C57BL/6-Tg(ACTB- EGFP)10sb/J. mOEC-GFP were cultured in Dulbecco's Modified Eagle

Medium/Nutrient F-12 (DMEM/F12) supplemented with 10% fetal bovine serum (FBS, Bovogen) and 10 ng/mL gentamicin (Life Technologies). The OECs were maintained in a humidified incubator at 37°C and 5% C0 2 . Three-dimensional cell cultures of olfactory ensheathing cells (OECs) were grown in liquid marbles

("spheroids"). The spheroids were transferred into an 8-well chamber with a final media volume of 200 μΐ ^ .

[0087] A comparative study between the 3D-tip and the commonly used 50/50 media exchange method with normal tips was performed.

[0088] Using the prototype 3D-tip, all 200 μΐ ^ of media in each well was aspirated from the cell culture and 200 μΐ ^ of fresh media was added to each of the 8 wells. Spheroids were imaged before and after media change using an Olympus (RTM) 1X73 microscope and analysis was performed using cellSens (RTM) imaging software from Olympus (RTM). The spheroids were maintained in culture for 7 days without media change for contamination assessment.

[0089] To perform the 50/50 media change, using a P200 Pipette and a normal tip, half of the 200 μΐ ^ of cell medium (100 μί) was carefully removed from each well in the 8-well chamber. 100 μΐ ^ of fresh medium was then added into each. Spheroids were imaged before and after media change using the Olympus (RTM) 1X73 microscope and analysis were performed using the cellSens (RTM) imaging software from Olympus (RTM).

[0090] Images taken of the spheroids before and after media change using each of the 3D-tip and the 50/50 method are shown in Figure 7. Figure 8 shows the percentage of spheroids remaining after media change using the 3D-tip and the 50/50 media exchange method. The scale bar marked with the asterisk represents 200 μιη.

[0091] The above demonstrated that the 3D-tip allowed complete media change whereas only half of the media could be successfully replaced with the 50/50 media exchange method using a conventional pipette tip. [0092] In addition, as shown in Figures 7 and 8, it was observed that on average 86 % of spheroids remained in the chamber after changing the media using the 3D-tip, whereas only 45 % of spheroids remained using the 50/50 media exchange method. This demonstrated that the conventional 50/50 media exchange technique caused a far greater loss of spheroids during aspiration compared to the new 3D-tip. In other words, the percentage of spheroids lost during the media change procedure using the 3D-tip significantly decreased in comparison with the 50/50 media exchange method using normal tips (p=0.0158).

[0093] In addition, no bacterial contamination was observed after changing the media using the 3D-tip.

Example 2 - Demonstrating the suitability of the 3D-tipfor immunofluorescence microscopy

[0094] To determine whether the 3D-tip is suitable for handling 3D spheroids during immunofluorescence assays, 3D co-cultures were performed of (1) astrocytes with SlOOp-DsRed Schwann cells (SC) and (2) astrocytes with SlOOp-DsRed olfactory ensheathing cells (OEC) in liquid marbles.

[0095] The medium was aspirated using a P200 Pipette and the 3D-tip. 3D co- cultures of (1) astrocytes with Schwann cells (SC) and (2) astrocytes with olfactory ensheathing cells (OEC) were fixed in 4 % PFA for 24 h at 4°C before being washed three times with phosphate buffered saline (PBS) using a P200 Pipette and the 3D-tip. Spheroids were then treated with 0.2 % Triton X-100 and 2 % bovine serum albumin (BSA) for 1 h, at RT and washed three times with PBS. PBS was aspirated and Goat anti-GFAP antibodies (1:500 in PBS) were added. The 8-well chamber was incubated at RT for 3 h with continuous shaking. Spheroids were washed three times with PBS. Secondary antibody Alexa Fluor (RTM) 488 Donkey anti-goat (1:500 in PBS) was added for lh at RT with continuous shaking. 3D spheroids were washed three times with PBS and stained with Hoechst (1:2000) for 15 min at RT. Spheroids were washed three times with PBS and the chamber was stored in the dark at 4°C. Spheroids were imaged using an Olympus (RTM) confocal FVIOOO microscope.

[0096] Figure 9 shows images taken by the Olympus (RTM) confocal FVIOOO microscope of co-culture of astrocytes detected by anti-GFAP (green) and SIOOP- DsRed Schwann cells (red). Figure 10 shows images taken by the Olympus (RTM) confocal FVIOOO microscope of co-culture of astrocytes detected by anti-GFAP (green) and SlOOP-DsRed OEC (red). The Schwann cells and the OEC cells were both stained with Hoechst (shown in blue in Figures 9 and 10). In both of Figures 9 and 10, the white scale bar represents 100 μιη.

[0097] As shown in Figures 9 and 10, a specific detection of GFAP in astrocytes was observed. A specific detection of nuclei by Hoechst stain was also observed as well as a low background signal.

[0098] The accurate immunofluorescence and nuclear staining, together with the intact morphology of the 3D cell structures, show that the 3D-tips are suitable for performing assay and wash steps during immunofluorescence assays without damaging the spheroids.

Example 3 - Comparison between the 3D-tip and centrifugation for preserved (fixed) multicellular spheroid immunofluorescence microscopy

[0099] A comparative study between using the 3D-tip and using a conventional centrifugation method for immunofluorescence microscopy was performed using preserved (fixed) multicellular spheroids.

[0100] For assessment of the 3D-tip, 3D OEC spheroids were fixed using 4 % PFA for 24 h at 4°C. The spheroids were then washed three times with PBS using a P200 Pipette and the 3D-tip. 3D spheroids were then stained with Hoechst (1:2000) and CellMask Deep Red (1:2000) in PBS for 20 min at RT with continuous shaking.

Spheroids were washed three times with PBS using the 3D-tip and then stored in the dark at 4°C. Spheroids were imaged using the Olympus (RTM) 1X73 microscope before and after staining. The number of spheroids was determined using the AnaSP software.

[0101] For comparative assessment of conventional centrifugation, 3D OEC spheroids were fixed using 4 % PFA for 24 h at 4°C. The spheroids were then transferred into a 1.5 ml Eppendorf, centrifuged for 5 min at 1000 rpm and the supernatant was aspirated using a P200 Pipette and a normal tip. 3D cell spheroids were washed three times with PBS using the centrifugation method (5 min at 1000 rpm) previously described. Spheroids were then stained with Hoechst (1:2000) and CellMask Deep Red (1:2000) in PBS for 20 min at RT with continuous shaking. The spheroids were centrifuged for 5 min at 1000 rpm and the supernatant was aspirated using a P200 Pipette and a normal tip. Spheroids were washed three times with PBS using the centrifugation method (5 min at 1000 rpm) as previously and then stored in the dark at 4°C. Spheroids were imaged using the Olympus (RTM) 1X73 microscope before and after staining. The number of spheroids was determined using the AnaSP software.

[0102] Figures 11 to 13 comparatively illustrate the differences between using the novel 3D-tip and using conventional centrifugation methods for immunofluorescence assay of preserved (fixed) 3D OEC cultures. In particular, Figure 11 shows

representative images of fixed 3D OEC spheroids stained with Hoechst (blue) and CellMask Deep Red (red) using the 3D-tip and the centrifugation methods (using a Olympus (RTM) 1X73 microscope). The white scale bar provided in Figure 11 is representative of 50 μιη. Figure 12 illustrates the comparative sphericity of 3D structures after immunofluorescence assay using the 3D-tip and the centrifugation methods. Figure 13 comparatively illustrates the percentage of spheroids remaining after immunofluorescence assay using the 3D-tip and the centrifugation methods.

[0103] As evidenced by Figures 11 to 13, it was observed that the sphericity of the 3D structures was maintained using both the 3D-tip and the centrifugation method (Figures 11 and 12). However, 88 % of spheroids remained using the 3D-tip while, in contrast, only 66 % of spheroids remained using the centrifugation method (Fig. 11 and 13). [0104] Consequently, the percentage of spheroids lost using the 3D-tip significantly decreased in comparison with the centrifugation method (p= 0.0234).

[0105] In addition, the centrifugation step with three washes required 20 minutes whereas the use of the 3D-tip required less than one minute for the wash steps. Thus the use of the 3D-tip reduces the time needed for assays.

Example 4 - Comparison between the 3D-tip and centrifugation for fluorescent labelling of live (unfixed) multicellular spheroids

[0106] A comparative study between using the 3D-tip and using a conventional centrifugation method for fluorescence labelling of live (unfixed) multicellular spheroids was performed.

[0107] For assessment of the 3D-tip, 3D spheroids were stained with Hoechst (1:2000) and CellMask Deep Red (1:2000) in culture media for 20 min at 37°C, 5 % C0 2 . The spheroids were then washed three times with PBS using the 3D-tip and then stored in the dark at 4°C. Spheroids were imaged before and after the assay using the Olympus (RTM) 1X73 microscope. The number of spheroids was determined using the AnaSP software.

[0108] For comparative assessment of conventional centrifugation, 3D OEC spheroids were stained with Hoechst (1:2000) and CellMask Deep Red (1:2000) in culture media for 20 min at 37oC, 5 % C02. The spheroids were then transferred into a 1.5 ml Eppendorf, centrifuged for 5 min at 1000 rpm and the supernatant was aspirated using a P200 Pipette and a normal tip. Spheroids were washed three times with PBS using the centrifugation method (5 min at 1000 rpm) as described above and then stored in the dark at 4°C. Spheroids were imaged using the Olympus (RTM) 1X73 microscope before and after staining. The number of spheroids was determined using the AnaSP software. [0109] Figures 14 to 16 comparatively illustrate the differences between using the novel 3D-tip and conventional centrifugation methods for immunofluorescence assay of live (unfixed) 3D OEC cultures. In particular, Figure 14 shows representative images of unfixed 3D OEC spheroids stained with Hoechst (blue) and CellMask Deep Red (red) using the 3D-tip and the centrifugation methods. The white scale bar provided in Figure 14 is representative of 50 μιη. Figure 15 illustrates the comparative sphericity of 3D structures after immunofluorescence assay using the 3D-tip and the centrifugation methods. Figure 16 comparatively illustrates the percentage of spheroids remaining after immunofluorescence assay using the 3D-tip and the centrifugation methods.

[0110] The shape of spheroids was significantly altered with the centrifugation method whereas the sphericity of the 3D structures was maintained using the 3D-tip (p= 0.0265) (Figures 14 and 15). We also observed that 85 % of spheroids remained using the 3D-tip while, in contrast, only 36 % of spheroids remained using the centrifugation method (Figures 14 and 16). Consequently, the percentage of spheroids lost using the 3D-tips significantly decreased in comparison with the centrifugation method (p= 0.0018).

Suitability of 3D-tip for immunofluorescence microscopy of pancreatic cancer cells (BxPC-3) and neural progenitor cells (ReNcell VM)

[0111] To determine the suitability of the 3D-tip for handling various cell types, 3D cultures were performed of pancreatic cancer cells (BxPC-3) and neural progenitor cells (ReNcell VM). Pancreatic cancer cells (BxPC-3) and neural progenitor cells (ReNcell VM) were maintained in DMEM supplemented with 10% FBS and 10 ng/mL gentamicin and in DMEM/F12 supplemented with 10% fetal bovine serum, 1% N-2 Supplement (Life Technologies) and 10 ng/mL gentamicin, respectively. All the cells were maintained in a humidified incubator at 37°C and 5% C0 2 .

[0112] Like the OECs discussed above, 3D cultures of BxPC-3 and ReNcell VM were grown in liquid marbles ("spheroids"). The BxPC-3 and ReNcell VM spheroids were fixed and stained with Alexa Fluor™ 488 Phalloidin, to visualize actin filaments (F- actin), and with Hoechst to stain the nuclei. The BxPC-3 and ReNcell spheroids were fixed using 3% PFA for 24 hrs at 4°C, then permeabilised by 0.3% Triton-X 100 for 60 min at RT with continuous shaking. Then spheroids were stained with Alexa Fluor™ 488 Phalloidin (1: 1600) in PBS for 60 min at RT with continuous shaking. Spheroids were washed three times with PBS using the 3D-tip and then stored in the dark at 4°C.

[0113] Figures 17 and 18 shows representative images of fixed 3D BxPC-3 spheroids (Figure 17) and ReNcell VM spheroids (Figure 18) fixed stained with Alexa Fluor™ 488 Phalloidin (green) and Hoechst (blue) using the 3D-tip (using an Olympus FV3000 confocal laser scanning microscope). The white scale bar provided in each of Figures 17 and 18 represents 50 μιη.

[0114] Figures 17 and 18 show that a regular F-actin arrangement around each nucleus in the 3D cultures of is present both in the BxPC-3 spheroid and the ReNcell VM spheroid. Thus, the 3D-tip can be used for removing fluid from cell cultures comprising different types of cells, such as cancer cells, progenitor cells and stem cells.

[0115] It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.